鼠李唐乳杆菌对酒精性肝损伤的免疫调节作用

Immunomodulatory effects of Lactobacillus rhamnosus Gorbach Goldin on alcoholic liver injury

目的研究鼠李唐乳杆菌（LGG）对酒精性肝损伤Treg／Th17免疫调节作用及TLR通路的表达变化，为酒精性肝损伤早期治疗提供依据。方法10周龄雄性C57／BL6小鼠根据随机数字表均分为对照组、乙醇组和LGG＋乙醇组，每组6只。乙醇组小鼠用含有5％乙醇的Lieber-DeCarli液体饲料喂养10d，第11天清晨按小鼠体质量5g／kg予40％乙醇灌胃9h后处死；对照组小鼠用不含乙醇的Lieber-DeCarli液体饲料喂养相同时间后予5％麦芽糖灌胃9h后处死；LGG＋乙醇组小鼠用LGG菌液（LGG〉1mL／d）加含5％乙醇的Lieber-DeCarli液体饲料喂养相同时间后，予40％乙醇灌胃9h后处死。收集各组小鼠肝组织行HE和油红O染色，观察组织病理改变。检测各组小鼠血清ALT、AST含量。RT—PCR检测各组小鼠肠道紧密连接蛋白ZO-1、occludin和claudin-1 mRNA表达。Western免疫印迹法检测各组小鼠肝脏TLR4、髓样分化因子88（MyD88）、IL-1受体相关激酶（IRAK-1）、核因子-κB和TNF-α蛋白表达。流式细胞仪分析LGG对小鼠脾脏Treg／Th17平衡的影响。各组间的统计分析用单因素方差分析和多重比较分析。结果HE染色和油红0染色示，经LGG干预后小鼠肝脏内脂滴数量和大小明显改善。乙醇组ALT为（147．0±42．1）U／L，AST为（373．5±169．7）U／L，对照组分别为（56．8±15．3）U／L和（211．5±57．8）U／L，LGG＋乙醇组分别为（75．3±150．2）U／L和（211．7±36．3）U／L，各组间相比差异均有统计学意义（F值分别为19．474和4．702，P值分别为0．000和0．026）。乙醇组肠道紧密连接蛋白ZO-1、occludin和claudin-1mRNA表达明显低于对照组，LGG＋乙醇组紧密连接蛋白表达增加（F值分别为149．219、145．561和154．237，均P=0．000）。乙醇组小鼠肝脏内大肠埃希菌蛋白表达高于对照组，LGG＋乙醇组其表达减少（F=130．317，P=0．000）。乙醇组小鼠肝脏内TLR4、MyD88、IRAK-1、核因子-κB和TNF-α蛋白表达明显高于对照组，LGG＋乙醇组TLR4通路中各蛋白表达明显抑制。乙醇组小鼠脾脏中Treg所占比例明显低于对照组，LGG＋乙醇组小鼠Treg所占比例升高。乙醇组小鼠脾脏中Th17所占比例明显高于对照组，LGG＋乙醇组小鼠Th17所占比例降低。结论LGG对酒精性肝损伤有保护作用，能够调节Treg／Th17平衡，抑制TLR4通路激活，为酒精性肝损伤早期治疗提供依据。

Objective To study the effects of Lactobacillus rharnnosus Gorbach Goldin （LGG） on the balance of regulatory cell （Treg）/T helper cell 17 （Th17） and the expression changes of Toll-like receptor （TLR） signaling pathway in alcoholic liver injury. Methods After an acclimatizing period, 10- week male C57/BL6 mice were randomized into control group, alcohol group and treatment group with six mice in each group. The mice in alcohol group were fed with 5 % alcohol-containing Lieber-DeCarli diet for 10 days, followed by gavage of a single dose of 40%alcohol at 5g/kg by weight. The mice in control group were pair-fed the control isocaloric diets in which ethanol was replaced with maltose-dextrin; the mice in treatment group were given LGG in the liquid diet in which one mouse consumed 1 mL/d LGG. Blood andtissue samples were collected for stained with hematoxylin-eosin （HE） or Oil red O. Serum levels of alanine aminotransferase （ALT） and aspartate aminotransferase fAST） were detected, mRNA levels of ZO-1, claudin-1, and occludin were analyzed by real-time polymerase chain reaction （RT-PCR）. The expressions of TLR4, myeloid differentiation factor 88 （MyD88）, interleukin-1 receptor-associated kinase- 1 （IRAK-1）, nuclear factor （NF）-κB, and tumor necrosis factor （TNF）-α were analyzed by Western blot. Flow cytometry was used to analyze the balance of Treg/Th17 celIs in the spleen. One-way analysis of variance （ANOVA） and multiple comparison analysis were performed for multi-groups comparison. Results Compared with control group, HE and Oil red O staining demonstrated that LGG supplementation significantly reduced the number of lipid droplets. Serum ALT and AST levels in alcohol group （[147.0±42.1] U/L and [373. 5± 169. 7] U/L, respectively） were more elevated than control group （[56.8±15.3] U/L and [211.5±57.8] U/L, respectively）, and LGG supplementation decreased the serum levels of ALT and AST （[75.3 ± 150.2] U/L and [211.7 ± 36. 3] U/L, respectively） with statistical significant difference （F= 19. 474 and 4. 702, respectively; P=0. 000 and 0. 026, respectively）. The mRNA expression of tight junction proteins including ZO-1, occludin and claudin-1 were significantly decreased by alcohol feeding, which were reversed by LGG supplementation （F= 149. 219,145. 561 and 154. 237, respectively; all P = 0. 000）. The hepatic protein level of Escherichia coli in the LGG supplementation group was decreased compared with alcohol group （F= 130. 317, P= 0. 000）. The expressions of critical proteins such as TLR4, MyD88, IRAK-1, NF-κB, and TNF-α in the TLR4 signaling pathway in alcohol group were higher than the control group. The changes were reversed by the supplementation of LGG. The number of Treg was decreased by alcohol exposure, which was ameliorated by the supplementation of LGG administration. The number of Thl7 was increased by alcohol exposure, while supplementation of LGG administration decreased Th17 counts. Conclusion LGG is effective in protecting alcohol-induced hepatic injury, by balancing Treg/Th17 and inhibiting the upregulation of TLR4 signaling pathway, which could be a potential therapeutic target in alcohol liver disease.